Protection against ricin intoxication in vivo by anti-idiotype vaccination.

A BALB/c murine anti-ricin monoclonal antibody (mAb BG11-G2, IgG1K), was recently isolated and shown to passively protect syngeneic mice against ricin intoxication in vivo. New Zealand White rabbit polyclonal anti-idiotype (anti-Id) antibodies were raised to BG11-G2 anti-ricin mAb, and rendered specific by repeated absorption over agarose normal mouse immunoglobulin (Ig). The absorbed rabbit anti-Id antibodies lost reactivity to normal mouse Ig and to a BALB/c anti-T-2 mycotoxin IgG1K mAb (HD11), but remained reactive with BG11-G2 anti-ricin mAb. The rabbit anti-Id inhibited the binding of BG11-G2 mAb to ricin-coated wells, and elicited a specific and protective anti-ricin antibody response in naive BALB/c mice. Whereas all mice vaccinated with a control rabbit anti-Id antibody preparation died from in vivo ricin challenges, mice immunized with the rabbit anti-Id specific for BG11-G2 mAb were protected to various degrees. All mice vaccinated with rabbit anti-Id to BG11-G2 and challenged with ricin doses of 35 and 50 micrograms kg-1 body weight died from the challenge; however, a delay in the elapsed time between ricin administration and death was observed in these mice as compared to that of the control anti-Id-immune mice. Five of seven mice vaccinated with the rabbit anti-Id to BG11-G2 and subsequently challenged in vivo with a ricin dose of 20 micrograms kg-1 body weight survived the lethal in vivo ricin challenge, whereas all the control mice died.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of ricin by colorimetric and chemiluminescence ELISA.

A highly sensitive and specific ELISA was developed to detect ricin in biological fluids. The assay utilizes an affinity-purified goat polyclonal antibody to adsorb ricin from solution. The same antibody (biotinylated) is then used to form a sandwich, and avidin-linked alkaline phosphatase allows color development and measurement of optical density at 405 nm. Our routine assay uses a standard curve over the range of 0-10 ng/ml ricin, with accurate quantitation below 1 ng/ml (100 pg/well) in assay buffer as well as in a 1:10 dilution of human urine or 1:50 dilution of human serum spiked with ricin. Ricin measured in spiked samples demonstrated accuracy typically within 5% of the expected value in all matrices. The coefficient of variation ranged from 3-10% at 10 ng/ml to 8-25% at 2.5 ng/ml. Two variations on the routine assay were also investigated. First, lengthened incubation times and additional time for color development allowed accurate quantitation in serum dilutions as low as 1:2. Second, increased concentrations of biotinylated antibody and avidin-linked enzyme from 1:250 to 1:70 enhanced the sensitivity of the assay 10-fold, achieving a detection limit of at least 100 pg/ml (10 pg/well). The assay was also configured to a format based upon chemiluminescence, which allowed quantitation in the 0.1-1 ng/ml range, but was subject to slightly greater variability than the colorimetric assay.

Reversal of saxitoxin-induced cardiorespiratory failure by a burro-raised alpha-STX antibody and oxygen therapy.

Reversal of saxitoxin (STX; 10 micrograms/kg, ip) induced cardiorespiratory effects by oxygen ventilation and burro-raised alpha-STX antitoxin (60 mg/kg, i.v.) was studied in urethane-anesthetized guinea pigs acutely instrumented for concurrent monitoring of medullary respiratory-related single units, diaphragm EMG, Lead II electrocardiogram, arterial blood pressure (BP), arterial pH, and O2/CO2 tensions, electrocorticogram (ECoG), and end-tidal CO2. STX-induced cardiorespiratory effects included (1) a state of progressive bradypnea and hypercapnia; (2) a functional blockade of the diaphragm; (3) a prolongation of respiratory cycle duration; (4) an aberrant bulbar respiratory-related neuronal activity pattern; and (5) a decline in BP and heart rate. The therapeutic effect of artificial ventilation following STX-induced apnea was equivocal in that the cardiorespiratory activities, be they of central or peripheral nature, remained dysfunctional despite continued oxygen ventilation. Spontaneous breathing and cardiovascular performance following STX-induced apnea could all be promptly restored (typically in less than a minute) by combined oxygen/antitoxin therapy. Notable also was a state of uncompensated acidemia (as revealed by changes in arterial pH and CO2 tension) which persisted throughout the course of therapeutic intervention. Notwithstanding, the ventilatory frequency continued to be low, the central respiratory activity pattern remained aberrant, and the ECoG amplitudes were still depressed. In consideration of these findings, and of the large molecular weight of alpha-STX antitoxin (> 150,000 Da) which limits its entry into the CNS, we are of the opinion that the therapeutic effects of antitoxin are probably confined primarily to the periphery.

Polyclonal anti-idiotypes induce specific anti-saxitoxin antibody responses.

Polyclonal BALB/C mouse and New Zealand White rabbit anti-idiotypic antibodies were raised by immunization with a protein G-purified burro anti-saxitoxin IgG antibody preparation. Following absorption of non-anti-idiotype reactivity, murine and rabbit IgG were purified by protein A chromatography and used to immunize BALB/C mice for the induction of anti-saxitoxin antibody responses. Unconjugated BALB/C anti-idiotypes did not induce significant anti-saxitoxin reactivity in BALB/C mice, even after repeated immunizations. However, BALB/C mice immunized with purified BALB/C anti-idiotypes conjugated to keyhole limpet hemocyanin, or with purified, unconjugated rabbit anti-idiotypes, as aluminum hydroxide precipitates, induced significant and specific anti-saxitoxin immune responses. Saxitoxin, a sodium channel blocker, can protect cells treated with veratridine and ouabain, whose respective actions are to open sodium channels and to block the activity of Na/K-ATPase. The anti-idiotype-induced anti-saxitoxin antibodies inhibited saxitoxin from protecting against cell death induced by veratridine and ouabain treatment. These and other published experimental results strengthen the concept of anti-idiotype-based vaccines in eliciting protective immunity against a variety of low molecular weight, nonproteinaceous biological and chemical toxins, whose extreme toxicity does not allow their use as safe immunogens.

Polyclonal anti-idiotypes induce antibody responses protective against ricin cytotoxicity.

Protein G-purified goat anti-ricin IgG, previously demonstrated to protect against ricin toxicity in vitro and in vivo, was used to raise BALB/c mouse and New Zealand White rabbit polyclonal anti-idiotypic antibodies. The generated anti-idiotypic sera were repeatedly absorbed over agarose conjugated to normal goat immunoglobulins, and purified by protein A-agarose affinity chromatography. Immunization of BALB/c mice with BALB/c anti-idiotypes did not result in a significant anti-ricin antibody response. However, injection of BALB/c mice with BALB/c anti-idiotypes conjugated to keyhole limpet haemocyanin (KLH) or with unconjugated rabbit anti-idiotypes resulted in specific and anti-ricin immune responses. The anti-idiotype-induced anti-ricin antibody responses protected against the in vitro cytotoxicity of ricin, a potent plant-derived protein synthesis inhibitor, as assessed by the murine EL-4 leukaemia cell assays. Thus, anti-idiotype-based vaccines may represent an alternative, safe and effective means of inducing protective immunity against toxins such as ricin, whose extreme in vivo toxicity render them unsafe as immunogens.

Monoclonal antibody prophylaxis against the in vivo toxicity of ricin in mice.

A BALB/c murine IgG1 monoclonal antibody, designated BG11-G2, specific for ricin was generated. BG11-G2 antibody did not bind to either purified ricin chain A or chain B, but recognized an antigenic determinant whose conformation requires the combination of the two chains in the formation of the native ricin molecule. It did not react with the protein synthesis inhibitor, T-2 mycotoxin, or with the sodium channel blockers, saxitoxin and tetrodotoxin. As little as 0.78 micrograms/ml of BG11-G2 IgG1 anti-ricin monoclonal antibody completely protected against the in vitro toxicity of ricin as determined by [3H]leucine uptake in EL-4 cell assays. Passive intraperitoneal infusion of purified BG11-G2 antibody into BALB/c mice one day prior to a lethal challenge with ricin considerably delayed the onset of toxicity and death. Better protection was obtained with BG11-G2 infused before and after ricin challenge.

Protection of mice from inhaled ricin by vaccination with ricin or by passive treatment with heterologous antibody.

Mice were vaccinated subcutaneously with 25 micrograms kg-1 of ricin in the presence of Freund's complete adjuvant or Ribi adjuvant, followed by a boost 14 days later with 50 micrograms kg-1 ricin in Freund's incomplete adjuvant or Ribi adjuvant, respectively. Three subsequent boosts at 28-day intervals with 25 micrograms kg-1 ricin yielded high anti-ricin antibody titres as determined by ELISA. Vaccinated animals were exposed to an aerosolized LD99 dose of ricin. With the exception of one death not attributable to ricin intoxication, all vaccinated mice survived the lethal aerosol exposure. In addition, a passive protection regimen was evaluated in mice pretreated with 100 micrograms purified goat anti-ricin IgG administered intravenously, and then challenged with ricin intravenously. All were resistant to 125 micrograms kg-1 of ricin, a dose greater than 25 times the intravenous lethal dose. Mice injected intravenously with 5 mg of the same IgG were protected from a lethal aerosol challenge. These results indicated that it is possible to protect animals from inhaled ricin by vaccination or passive administration of specific antibodies.

Anti-idiotype vaccines in toxicology.

The majority of naturally occurring biological and chemical toxins are highly lethal, nonproteinaceous, low molecular weight substances which exert their toxicity through a variety of mechanisms. Their relative small size and extreme in vivo toxicity have hampered the development of protective vaccines. We have investigated the feasibility of anti-idiotype-based vaccines which utilize antibodies for inducing a systemic and protective immunity against the in vivo toxicity of some of these toxic substances. A murine IgG1 monoclonal anti-T-2 mycotoxin antibody protective against mycotoxin toxicity was generated. This antibody was used to produce a second generation monoclonal anti-idiotype antibody which was capable of serologically mimicking the tertiary conformation of the nominal antigen, i.e., T-2 mycotoxin. Administration of the monoclonal anti-idiotype antibody to mice induced a circulating and protective antibody response against the in vitro and in vivo toxicity of T-2 mycotoxin. Antibody-based vaccines may represent the only safe and effective strategy for the design of protective vaccines against small nonproteinaceous toxic compounds whose extreme toxicity prevents their use as safe immunogens. The potential of antibody-based vaccines for producing protective immunity against low molecular weight chemical and biological toxins is discussed.

Anti-idiotype-based vaccines against biological toxins.

Biological toxins produced by living organisms represent one of the major sources of contamination of stored grain and agricultural products, and other food sources. The majority of these biological toxins are highly lethal, nonproteinaceous low-molecular-weight chemical compounds which exert their potent toxicity through a variety of mechanisms. Because of their small size, they generally do not induce a significantly high affinity protective antibody response upon toxin exposure, even when conjugated to large protein carriers which enhance their immunogenicity. Moreover, the very toxic nature of biological toxins precludes their use as immunogens in the induction of protective immunity. To circumvent this difficulty, an attempt was made to develop antibody (anti-idiotype)-based vaccines against a protein synthesis inhibitor, the trichothecene mycotoxin T-2, and the sodium channel blockers tetrodotoxin and saxitoxin. Protective monoclonal antitoxin antibodies were first generated and then used to induce specific monoclonal anti-idiotype antibodies. Specific anti-idiotype antibodies were assessed for their ability to induce in vivo protective immunity against toxicity.

Structure/function studies of T-2 mycotoxin with a monoclonal antibody.

A BALB/C murine monoclonal antibody (mAb) specific for the trichothecene mycotoxin T-2 was previously generated. The anti-T-2 antibody, designated HD11, can detect T-2 in the nanogram range employing an enzyme-linked immuno-sorbent assay. The HD11 antibody at a concentration of 1 microgram/ml completely protected against the T-2-induced cytotoxicity of the human epidermoid carcinoma cell lines Hep-2 and KB. Fine specificity analysis was performed using 10 structurally related T-2 metabolites to inhibit the specific binding of HD11 to T-2 mycotoxin. The results suggest a binding specificity of the protective HD11 antibody for the bulky hydrophobic alkyl side chain at position R5 and the acetyl groups at positions R2 and R3 of the T-2 mycotoxin molecule. HD11 anti-T-2 mAb, which bound to the T-2 metabolite acetyl T-2 (with a substituted acetyl group at R1 position), efficiently neutralized its in vitro cytotoxicity. On the other hand, the cytotoxicity of T-2 metabolites neosalaniol and 3' OH HT-2, both of which lack the alkyl side chain at position R5 and which did not bind to HD11, was unaffected by HD11.

Monoclonal anti-idiotype induces protection against the cytotoxicity of the trichothecene mycotoxin T-2.

An IgG1 mAb, designated HD11, specific for the trichothecene mycotoxin T-2 and capable of neutralizing its cytotoxicity was used to generate a syngeneic monoclonal anti-Id antibody. The generated anti-Id mAb, designated DE8, specifically bound to HD11 anti-T-2 mAb, and not to IgG1 mAb of irrelevant specificity or to normal mouse Ig. DE8 inhibited the binding of HD11 anti-T-2 to T-2-BSA-coated plates, whereas a control anti-Id mAb did not, suggesting recognition of an Id determinant associated with the T-2 binding site of HD11. Moreover, the binding of HD11 to DE8 and that of DE8 to HD11 were specifically inhibited by free T-2 mycotoxin. DE8 mAb was efficient in abrogating the protective effect of HD11 in the cytotoxicity of T-2 on the human epidermoid carcinoma cell line Hep-2. In vivo immunization of BALB/c mice with DE8 conjugated to KLH induced an anti-T-2 antibody titer comparable to that obtained with T-2-OVA immunization, whereas immunization with unconjugated DE8 resulted in a lower titered anti-T-2 response. Immunization with DE8-keyhole limpet or with unconjugated DE8 induced anti-T-2 antibody responses characterized by expression of "HD11-like" Id and by protection against T-2 cytotoxicity. However, the T-2-OVA-induced anti-T-2 response lacked the HD11+ Id and was only partially protective against T-2 cytotoxicity. This represents the first demonstration of the use of an anti-Id based vaccine in the in vivo induction of a protective antibody response against the cytotoxicity of a nonproteinaceous, small m.w. biologic toxin, whose very toxic nature precludes its use as the immunogen.

Distribution and elimination of brevetoxin PbTx-3 in rats.

After i.v. administration, [3H]PbTx-3 was rapidly cleared from the blood; less than 10% remained after 1 min. Within 30 min, radiolabel distributed to skeletal muscle (69.5%), liver (18.0%), and intestinal tract (8.0%). Over 24 hr, radiolabel decreased in muscle, remained constant in liver, and increased in the intestinal tract and feces. Elimination occurred via feces (75.1%) and urine (14.4%), with 9.0% remaining in the carcass after 6 days. This distribution and elimination profile suggested that the liver was the major organ of metabolism and that biliary excretion was an important route of elimination. Thin-layer chromatography confirmed the presence of brevetoxin metabolites in fecal extracts. Skeletal muscle does not appear to be a site of metabolism, but a storage compartment, from which toxin is slowly released prior to clearance by the liver. These studies are the first demonstration of in vivo brevetoxin metabolism in mammals.

Anti-idiotypic antibodies against a monoclonal antibody specific for the trichothecene mycotoxin T-2.

A BALB/c murine monoclonal antibody against the trichothecene mycotoxin T-2 was generated. The antibody, designated HD11, specifically bound T-2 mycotoxin. The binding of HD11 to T-2 conjugated to bovine serum albumin was inhibited by free T-2 toxin but not by the water-soluble heterocyclic guanidines saxitoxin and tetrodotoxin. The T-2 detection limit in an enzyme-linked immunosorbent assay with HD11 was in the nanogram range. The in vitro cytotoxicity of T-2, as measured by the inhibition of radiolabeled leucine uptake of the human epidermoid carcinoma Hep-2 and KB cell lines, was completely reversed by the addition of HD11. Rabbit anti-idiotypic antibodies specific for HD11 were generated and characterized.

Detection and quantitation of T-2 mycotoxin in rat organs by radioimmunoassay.

A standard radioimmunoassay was compared with radiochromatography for the ability to detect unlabeled T-2 mycotoxin in organs from exposed animals. When 10% of HT-2, the only known metabolite that cross-reacts with T-2, was included and expressed as T-2 equivalents in the radiochromatographic detection, correlation between toxin detection in liver, spleen, and kidney by the 2 techniques was r = 0.98. An unknown metabolite was detected in heart extract by radiochromatography. Inclusion of this material in the T-2 equivalents detected by radiochromatography indicated a near-perfect correlation (r = 0.95; p greater than 0.05; slope = 0.82; y = intercept = 72) among all 4 tissues.

A rapid, large scale purification procedure for gibbon interleukin 2.

Interleukin 2 (IL 2) was purified from the conditioned medium of a gibbon T cell line, MLA144, which releases IL 2 constitutively. The IL 2 was obtained free of contaminating proteins by a simple process consisting of an initial batch purification on trimethylsilyl-controlled pore glass followed by reversed phase high pressure liquid chromatography. Overall recovery of IL 2 activity ranged from 70 to 100% of initial activity and yielded 2 X 10(6) or greater units of IL 2 per 15 liters of serum-free MLA144 conditioned medium. The specific activity of purified IL 2 ranged from 0.5 to 1 X 10(8) IL 2 U/mg protein. The purified IL 2 showed four molecular species when analyzed by two-dimensional isoelectric focusing-SDS-polyacrylamide gel electrophoresis. Each of the four molecular forms was active in the bioassay for IL 2 activity. Three molecular forms had apparent m.w. of 16,000 but different isoelectric points of 5.9, 6.3, and 6.7. One molecular form had an apparent m.w. of 15,000 and an isoelectric point of 7.2. The most abundant form of IL 2 had an apparent m.w. of 16,000 and an isoelectric point of 6.3. The purified IL 2 supported the growth of IL 2-dependent lymphocytes to a greater extent than did the same level of crude IL 2-containing MLA144 conditioned medium. The ability to purify large amounts of IL 2 by a rapid and efficient procedure will be of great help in both biochemical and immunologic studies of this lymphokine.

Discordant Epstein-Barr virus nuclear antigen (EBNA) antibody patterns in nasopharyngeal carcinoma.

Epstein-Barr virus nuclear antigen (EBNA) preparations from three sources were tested with sera from normal individuals and patients with Hodgkin's disease, breast carcinoma, Burkitt's lymphoma, and American and Chinese nasopharyngeal carcinoma. Individual sera with discordant antibody patterns were noted in all groups. Sera from both NPC groups gave significantly higher anti-EBNA titers on cell lines converted with P3HR-1 or B95-8 virus compared with anti-EBNA titers on Raji cells. Anti-EBNA titers of Chinese NPC sera showed no correlation among the three EBNA sources, while all other groups had highly correlated titers. Cross-absorption experiments present evidence for more than one antigenic determinant on EBNA. These results suggest an additional parameter for distinguishing Chinese NPC from other EBV-related disorders.

Sera from patients with undifferentiated nasopharyngeal carcinoma contain a factor which abrogates specific Epstein-Barr virus antigen-induced lymphocyte response.

A unique association of Epstein-Barr virus (EBV) with the undifferentiated nasopharyngeal carcinoma (NPC) is a well acknowledged phenomenon. We report here the detection of a factor present in the sera of NPC patients which inhibits the blastogenic response of lymphocytes from EBV seropositive individuals to EB virions or soluble antigens. This lymphocyte-stimulation inhibitor (LSI) was found to be associated with the IgA fraction of the serum immunoglobulins. No inhibitory activity was detected in the sera and their immunoglobulin fractions from healthy (both EBV-seropositive and seronegative) individuals and patients with other carcinomas of the head and neck region. Interestingly, the IgA-LSI was absent in the sera of NPC patients who were successfully treated and remained in remission, while it was readily detectable in the sera of NPC patients in relapse, LSI-positive IgA fractions did not inhibit mitogenic response of lymphocytes to phytohemagglutinin. Taken together, the data presented suggest that LSI is a specific inhibitor of the response of sensitized lymphocytes to EBV antigens and that it may indeed represent a marker of great clinical significance regarding undifferentiated nasopharyngeal carcinoma, particularly for its prognosis.

Infection of rhesus monkeys and chimpanzees with Epstein-Barr virus.

Seventy-two nonhuman primates were entered into a long-term study to evaluate the pathogenicity of Epstein-Barr virus (EBV). Infectious virus was inoculated into 42 rhesus monkeys, 4 chimpanzees and 1 cynomolgus monkey. Immunostimulation or immunosuppression was attempted in 34 of these animals to enhance the oncogenic potential of the virus. Eleven inoculated animals were followed for more than 3 years and two were observed for 8 years. No tumors were observed in any of the animals; however, serological evaluation of the 47 inoculated primates and 25 matched controls indicated that at least 14 rhesus monkeys and the cynomolgus monkey were successfully infected with EBV. The potential use of rhesus monkeys as a model for EBV-induced disease in humans is discussed.

Development of antibodies reactive in antibody-dependent cellular cytotoxicity in infectious mononucleosis.

Serial sera from patients with infectious mononucleosis were examined for the emergence of antibodies reactive in antibody-dependent cellular cytotoxicity tests, using Epstein-Barr virus-superinfected Raji cells as targets. For this specific purpose, the antibody-dependent cellular cytotoxicity test proved to be of limited sensitivity because only relatively high serum dilutions can be tested dependably, due to prozone effects at low serum concentrations, and because antibody-dependent cellular cytotoxicity reactions at the 5% level are not always statistically significant. Under the conditions of the test, antibody-dependent cellular cytotoxicity-reactive antibodies were not measurable, or only barely measurable, in early-acute-phase sera, but they became detectable during convalescence and increased thereafter, gradually over many months to the range of titers seen in healthy persons after long-past-primary Epstein-Barr virus infections. The percentages of antibody-dependent cellular cytotoxicity ultimately attained were on the order of 20% in most patients and healthy individuals, but in others did not exceed 10%. The likely identity of the antibodies reactive in the test with antibodies to late Epstein-Barr virus-determined cell membrane antigens has been discussed.

Infectious mononucleosis and the Epstein-Barr virus.

Recent elucidation of the relationship between the Epstein-Barr virus and infectious mononucleosis has resulted in the development of new diagnostic serological tests, and has amplified our knowledge of the epidemiological and clinical aspects of the disease. The history, epidemiology, clinical characteristics, diagnostic features, and therapy of infectious mononucleosis are reviewed. This is done in the light of recent knowledge concerning the Epstein-Barr virus as well as previous studies employing the traditional diagnostic criteria of heterophil positivity, the classical clinical symdromes, and characteristic changes in the blood cell count. Immunological studies concerning host resistance and its occasional failure are reviewed with particular reference to T and B lymphocyte activity in the disease.