In vitro correlate of immunity in a rabbit model of inhalational anthrax.

A serological correlate of vaccine-induced immunity was identified in the rabbit model of inhalational anthrax. Animals were inoculated intramuscularly at 0 and 4 weeks with varying doses of Anthrax Vaccine Adsorbed (AVA) ranging from a human dose to a 1:256 dilution in phosphate-buffered saline (PBS). At 6 and 10 weeks, both the quantitative anti-protective antigen (PA) IgG ELISA and the toxin-neutralizing antibody (TNA) assays were used to measure antibody levels to PA. Rabbits were aerosol-challenged at 10 weeks with a lethal dose (84-133 LD(50)) of Bacillus anthracis spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. Results showed that antibody levels to PA at both 6 and 10 weeks were significant (P<0.0001) predictors of survival.

In vitro correlate of immunity in an animal model of inhalational anthrax

The incidence of anthrax in humans is extremely low. Human vaccine efficacy studies for inhalational anthrax cannot be conducted. The identification of a correlate of protection that predicts vaccine efficacy is crucial for determining the immune status of immunized humans. This surrogate marker of immunity can only be established by using an appropriate animal model. Numerous studies showed that protective antigen (PA) is the principle protective antigen in naturally- or vaccine-induced immunity. However, attempts to correlate the quantity of anti-PA antibodies with protective immunity in the guinea pig model for anthrax and various vaccine formulations have failed. In these studies, we used the licensed anthrax vaccine adsorbed (AVA) in rabbits. Groups of New Zealand white rabbits, 10 or 20 per group, were immunized intramuscularly (two doses, 4 weeks apart) with varying doses of AVA, ranging from a human dose to 1:256 dilution in sterile phosphate-buffered saline (PBS). Control rabbits received PBS/Alhydrogel according to the same schedule. Each rabbit was bled 2 weeks after the second dose, and antibody levels to PA measured by both the quantitative anti-PA IgG ELISA and the toxin-neutralizing antibody (TNA) assay. Rabbits were aerosol-challenged 10 weeks from day 0 with a lethal dose of Ames spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. All the controls died. Rabbit survival was compared with the antibody response. Statistical models were used to test for significance of the peak antibody responses to predict survival. Results showed that both the amount of anti-PA IgG and TNA titres present in the sera at the time of the peak antibody response were significant (P < 0.0001) predictors of survival. These results demonstrate that the humoral immune response to AVA can predict protection in the rabbit model of inhalational anthrax.

Intranasal stimulation of long-lasting immunity against aerosol ricin challenge with ricin toxoid vaccine encapsulated in polymeric microspheres.

Intranasal (i.n.) immunization with ricin toxoid (RT) vaccine encapsulated in poly (lactide-co-glycolide) microspheres (RT-PLG-Ms) and poly (L-lactide) microspheres (RT-PLA-Ms) stimulated systemic and mucosal immune responses and protected mice from aerosolized ricin intoxication. High titers of anti-ricin IgG2a were stimulated in the serum of mice with one or two doses of RT-Ms 6 weeks postimmunization. However, in the lungs, no IgG2a or total IgG was elicited either with RT-Ms or with aqueous RT. At 6 weeks postimmunization, a single dose of the RT-Ms stimulated secretory IgA (sIgA) in the lungs of four of six mice, but a second immunizing dose did not enhance the stimulation. A single dose of aqueous RT vaccine failed to stimulate sIgA in the lungs, while, a second dose induced sIgA in 50% of the mice. One or two i.n. doses of RT-Ms protected most of the mice against lethal aerosol-delivered ricin toxin 6 weeks postimmunization. In contrast, protection was absent or marginal after one or two doses of aqueous RT vaccine. In both studies, the protection against lethal aerosol challenge was significantly better with one dose of RT-Ms than with two doses of aqueous vaccine, which may be attributed to the induction of sIgA in the lungs and the serum. Duration of the IgG2a and IgA in the serum, particularly that of IgG2a was much longer after the administration of RT-Ms than after the aqueous vaccine. The geometric mean IgG2a titers stimulated with two doses of RT-Ms remained high during 40 weeks postimmunization and were up to 25 times higher than the titers induced with aqueous RT vaccine. After 6 weeks, the IgG2a induced by two doses of aqueous vaccine was no longer detectable. Persistence of antibody response was predictive of efficacy. At 1 year postimmunization with two doses of RT-Ms, 100% of mice were protected against lethal ricin challenge. However, at the same time no protection was afforded by two doses of aqueous RT. The results of the present study consistently demonstrated the advantages of microencapsulated RT vaccine to stimulate effective and long-lasting protection by i.n. administration.

Dependence of ricin toxoid vaccine efficacy on the structure of poly(lactide-co-glycolide) microparticle carriers.

Biodegradable microparticles made of poly(lactide-co-glycolide) (PLG) were used for protracted and pulsed-release of the incorporated ricin toxoid (RT) vaccine to reduce the multiple immunization doses and the time required to induce complete protection against lethal aerosol-borne ricin challenge. The release rate of RT encapsulated in PLG microparticles was controlled by polymer selection and varying the preparation procedures, which allowed us to control microparticle size and the distribution of the vaccine in the polymeric matrix. PLG-microparticles in which RT vaccine was distributed heterogeneously in small pockets stimulated a rapid antibody response which was independent of the polymeric composition of the carriers. PLG-microparticles in which RT vaccine was distributed homogeneously throughout the polymeric matrix induced a slower antibody response, which depended on the polymeric composition of the carriers. Administration of RT in homogeneous microparticles made from 50/50 PLG or 100% polylactide stimulated two distinct anti-ricin IgG peaks, while RT in heterogeneous microparticles stimulated identical IgG peaks. An early (3 weeks) and long-lasting (1 year or longer) anti-ricin antibody response was evoked by a single administration of encapsulated RT vaccine when prepared by the above-mentioned conditions. In contrast, three administrations of the aqueous RT were required to stimulate similar antibody response. Reduction of immunization time from 6 to 4 weeks was achieved with RT encapsulated in small homogeneous microparticles but not with homogeneous large microparticles. These results demonstrated the usefulness of biodegradable microparticles to improve the efficacy of immunization with RT vaccine and probably many other vaccines as well.

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.