Macrophage-derived cell lines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin.

We present evidence that Bacillus anthracis lethal toxin (LT) suppresses rather than induces proinflammatory cytokine production in macrophages. Suppression is observed with extremely low levels of LT and involves inhibition of transcription of cytokine messenger RNA. Thus, LT may contribute to anthrax pathogenesis by suppressing the inflammatory response.

Idiotype cascades associated with the CD4-HIV glycoprotein 120 interaction: immunization with anti-idiotypic antibodies induces anti-anti-idiotypic responses with anti-CD4 specificity and in vitro neutralizing activity.

Anti-idiotypic antibodies (Ab-2), which were generated in baboons against a mouse monoclonal antibody specific for the CD4 molecule expressed on human T cells, were used to produce anti-anti-idiotypic antibodies (Ab-3) in mice. This response induced by Ab-2 immunization of BALB/c mice was classified as anti-anti-idiotype (Ab-3) on the basis of the ability of the mouse Ab-3 to (1) specifically bind the baboon Ab-2 preparation, but not irrelevant baboon IgG preparations, (2) inhibit the binding of the anti-CD4 Ab-1 preparation to the baboon Ab-2, and (3) recognize a second baboon Ab-2, along with a rabbit Ab-2 specific for the monoclonal anti-CD4 Ab-1 preparation. The murine Ab-3 response also recognizes the CD4 molecule expressed on a human CD4+ T cell line, as determined by flow cytometry; recognizes the same epitopes on the CD4 molecule as the Ab-1; inhibits HIV-1 syncytium formation; and neutralizes HIV-1 primary isolates in vitro. These studies suggest that Ab-3 responses can be induced by anti-Id immunization, which serologically mimicks the antigen and Id specificities of the monoclonal anti-CD4 preparation used to generate the anti-Id. Thus, the Ab-3 response exhibits the characteristics of a population that represents the internal image of the Ab-1.

The baboon as a nonhuman primate model for assessing the effects of maternal immunization with Haemophilus influenzae type b polysaccharide vaccines.

These studies were performed to assess the utility of the baboon as a nonhuman primate model to evaluate vaccines for use in humans. Specifically, we examined the antibody response of baboons immunized during the third trimester of pregnancy with Haemophilus influenzae type b (Hib) polyribosylribitol phosphate (PRP) conjugate and unconjugated polysaccharide vaccines. Some of the vaccinated mothers failed to respond to a single immunization with unconjugated Hib PRP. Specific Hib PRP immunoglobulin G (IgG) but not IgM antibodies cross the baboon placenta and are detected in the offspring. Higher-titer baboon anti-Hib PRP did not express two previously defined cross-reactive human anti-Hib PRP idiotypes and was biased towards lambda light-chain expression. Spectrotype analysis indicated that baboon anti-Hib PRP was restricted in heterogeneity and oligoclonal.

Purification and characterization of secretory IgA from baboon colostrum.

In this report, we describe a method for purifying secretory immunoglobulin A (sIgA) from baboon (Papio anubis) colostrum. The colostrum was first clarified by centrifugation and then analyzed with various anti-human Ig-specific immunologic reagents. Cross-reactive IgA in the baboon colostrum was identified by ELISA. Western blot analysis also demonstrated cross-reactive epitopes associated with human IgA1, IgA2, secretory component (SC), and joining (J) chain. To purify the sIgA, colostrum was separated into 4 distinct fractions by gel filtration chromatography. Analysis of the individual fractions by ELISA indicated that the IgA elutes over one peak. The IgA fraction was compared with purified human sIgA on SDS-PAGE, and exhibited heavy (H) chains, light (L) chains, SC, and J chain. The baboon colostrum was also analyzed by ELISA for specific IgG H and L chain epitopes utilizing monoclonal antibodies (MAbs). No significant quantity of IgG was detected in the baboon colostrum or in the individual 4 fractions, while L chain reactivity was observed in the sIgA fraction. The sIgA fraction was pooled, concentrated, and was found to contain approximately 7 mg/ml sIgA. To determine if the baboon sIgA was dimeric like human sIgA, the purified sIgA was sized by molecular sieve chromatography. The molecular size of the sIgA preparation (350 kDa) was determined empirically by comparison to known molecular species used to calibrate the column. In addition, native SDS-PAGE indicated that baboon sIgA, like human sIgA, migrates between IgG and IgM, suggesting it has a dimeric form. The purified baboon sIgA preparation should prove useful in the future study of mucosal immune responses induced in non-human primate species and for the generation of sIgA-specific immunological reagents.

Immunogenicity of group B Streptococcus type III polysaccharide-tetanus toxoid vaccine in baboons.

Maternal vaccination has been proposed as a rational approach for the prevention of neonatal group B streptococcal (GBS) disease. In this study, baboons were used as a nonhuman primate model to evaluate the immunogenicity of a GBS type III glycoconjugate vaccine. Type III-specific immunoglobulin G with opsonic activity was induced after vaccination with type III polysaccharide coupled to tetanus toxoid administered with an aluminum adjuvant. This suggests that baboons could be used in evaluating maternal transfer of GBS-specific antibodies by vaccination during pregnancy.

Serologic crossreactions among primate immunoglobulins.

We have generated and characterized 50 murine monoclonal antibodies (mAb) specific for baboon IgG. We examined crossreactivity of these mAb to baboon IgM and immunoglobulin (Ig) of various other primates including human, chimpanzee, rhesus monkey, cynomolgus monkey, and African green monkey. Those mAB that crossreacted with human IgG were further examined using myeloma proteins for specificity to human Ig subclasses. One mAB crossreacted with all four human IgG subclasses and with human IgM. We further analyzed this reactivity utilizing Bence Jones proteins representative of various light (L) chain germline gene family products. This mAB reacted with Bence Jones proteins indicating the recognition of a kappa (k) L chain specificity associated with the kappa I, kappa III, and kappa IV subgroups, but not with kappa II. Based on the differences between kappa II germ line gene encoded L chains and the other kappa L chain subgroups, we ascribe this reactivity to six amino acids that define a discontinuous epitope.

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)

Inhibition of retrovirus-induced syncytium formation by photoproducts of a brominated 1,8-naphthalimide compound.

A major disadvantage of conventional phototherapy is the requirement for the in situ delivery of stimulating photoenergy subsequent to the binding of photochemicals to target malignant cells, or virus-infected cells, or viruses. This drawback has resulted in considerable limitation in the use of photochemicals in photomedicine. To circumvent this problem, we have investigated the antiviral efficacy of a brominated 1,8-naphthalimide photocompound, termed LY66Br [3-bromo-4-(hexylamino)-N-hexyl-1,8-naphthalimide], which upon exposure to visible light at 420 nm generates independently of oxygen one or more stable antiviral molecular photoproducts (e.g., is 'preactivated'). Human cell lines infected with the human immunodeficiency virus type 1 (HIV-1), or with the human T-lymphotropic virus type-1 (HTLV-I) exposed to photochemical products of LY66Br (P-LY66Br) completely lost their ability to form syncytia in vitro. Photoproducts of P-LY66Br retain full antiviral activity for at least 3 and 6 weeks when stored at room temperature and at -80 degrees C, respectively. Concentrations of P-LY66Br, effective in inhibiting syncytium formation mediated by HIV-1 and HTLV-I, were nontoxic to normal red cell components of whole blood (red blood cell 2,3-diphosphoglyceric acid, adenosine triphosphate, osmotic fragility or blood type antigens). Additionally, no evidence of acute toxicity was demonstrated in mice following an intravenous bolus inoculation to achieve plasma concentration of 600 microM of P-LY66Br. These findings represent the first demonstration of inhibition of retrovirus-induced syncytium formation by a photochemical product, and justify further investigation of the preactivation process of photochemicals in the treatment of systemic viral infections such as the acquired immunodeficiency syndrome (AIDS), in cancer therapy, and in sterilization of banked blood products.

Production and characterization of murine monoclonal antibodies specific for baboon IgG heavy and light chain epitopes.

We have generated a panel of murine monoclonal antibodies (MAbs) that recognize baboon IgG epitopes. The reactivity of the MAbs with IgG from other primate species was also examined. Specificity for IgG heavy (H) or light (L) chain epitopes was determined by Western blot analysis. The H chain-specific MAbs were analyzed for IgG subclass specificity and the L chain-specific MAbs for reactivity with baboon IgM and polymeric sIgA. Finally, an ELISA was developed to demonstrate the utility of the MAbs in analysis of humoral immune responses in baboons.

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.

Neutralization of HIV-1 and inhibition of HIV-1-induced syncytia by 1,8-naphthalimide photoactive compound.

The antiviral property of a newly designed class of 1,8-naphthalimide photochemical compounds was investigated. One such photoactive compound, 1,14-bis-(N-hexyl-3'-bromo-1,8'-naphthalimide-4'-yl)-1,4,11,14- tetraazatetradecane-5,10-dione (diED66Br), when activated to an excited state by visible light (420 nm), effectively neutralized the in vitro infectivity of human immunodeficiency virus (HIV-1). Light-activated diED66Br also inhibited syncytium formation induced by cells infected with HIV-1. Nonactivated diED66Br was completely ineffective. The neutralizing and syncytium-inhibiting doses of activated diED66Br had no effect on normal human peripheral blood mononuclear cells. Radioimmunoprecipitation analysis indicated that diED66Br neutralizing activity resulted primarily from its ability to inhibit the binding of HIV-1 envelope glycoprotein gp120 to the CD4 cellular receptors. Although the exact molecular mechanism of viral neutralization by diED66Br has not been elucidated, its ability to neutralize HIV-1 infectivity and to inhibit syncytium formation supports further investigations of this photochemical as a potential therapeutic treatment of HIV-1 infection.

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.

HIV-1 neutralization and tumor cell proliferation inhibition in vitro by simplified analogues of pyrido[4,3,2-mn]thiazolo[5,4-b]acridine marine alkaloids.

The antiviral/antitumor marine alkaloid dercitin was used as a lead compound to design analogues with anti-HIV and tumor inhibitory activities. Deletion of structural features contributing to cytotoxicity led to analogues with lowered T-lymphocyte toxicity profiles. One compound, 5, induced complete protection against HIV-1 infectivity in vitro at 12.5 micrograms/mL (38 microM) without T-cell toxicity up to 400 micrograms/mL. Compound 4 and 5 also inhibited the binding of HIV-1 to H-9 lymphocytes. These compounds may exert antiviral activity by a unique dual extracellular and intracellular mode of action--both preventing viral attachment to lymphocytes as well as intercalating with viral nucleic acid. Analogues with higher cytotoxicity such as 2 which retain the thiazole ring of the natural product proved effective in completely inhibiting the cell proliferation of breast, colon, and lung tumor cell lines at 1.5 microM concentration compared to a 70 microM dose level of 5-fluorouracil. A means of molecular separation of antiviral activity from cytotoxicity was thus achieved, and putative pharmacophores for antiviral and antitumor actions of the prototype molecule dercitin have been deduced. The 2-thio-9-acridinone derivatives 4 and 5 represent a new structural type exhibiting activity against HIV in vitro, serving as chemical leads in the design of anti-AIDS agents, while thiazolo[5,4-b]acridines such as 2 provide leads in the drug design of new antitumor agents.

A comparative study of natural killer cell activity, lymphoproliferation, and cell phenotypes in nonhuman primates.

Three different species of nonhuman primates (baboons [Papio hamadryas], rhesus monkeys [Macaca mulatta], and African green monkeys [Cercopithecus aethiops]) were evaluated for their natural killer cell activity, and for the ability of their peripheral blood mononuclear cells to proliferate in response to known mitogens (concanavalin A, phytohemagglutinin, and pokeweed mitogen) and to react with a panel of mouse monoclonal antibodies directed against human leukocyte surface antigens. Rhesus monkeys displayed the highest natural killer cell cytotoxic activity (185.7 +/- 33 lytic units) compared with those of baboons (83.8 +/- 19 lytic units) and of African green monkeys from West Africa (39.08 +/- 8 lytic units) and from the Caribbean basin (37.9 +/- 9 lytic units). No correlation was observed between the natural killer cell cytotoxic activity and the percentage of CD16+ natural killer cells among the three species studied. High spontaneous proliferative capacity was observed in African green monkeys obtained from West Africa compared with those of the other species studied. Although no significant differences were noted in T and B cell mitogen-induced in vitro proliferation, baboon mononuclear cells were less responsive to concanavalin A (stimulation index of 16 +/- 3 [mean +/- standard error of mean]) than to phytohemagglutinin (stimulation index of 47 +/- 12). However, rhesus and African green monkey cells proliferated more efficiently in response to concanavalin A. Unlike in human beings where the ratio between helper-inducer (CD4+) and cytotoxic-suppressor (CD8+) T-lymphocytes is generally greater than 1, the CD4+/CD8+ ratios in baboons and rhesus and African green monkeys were 0.58, 0.69, and 0.35, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Preliminary studies of photoinactivation of human immunodeficiency virus in blood.

The transmission of human immunodeficiency virus (HIV) by blood or blood components is a major concern in blood banking. A photodynamic flow cell system was designed to inactivate cell-free HIV mixed with blood from a healthy donor. Blood containing 4 x 10(3) infectious units of HIV was treated with 10 and 20 micrograms per mL of commercially available dihematoporphyrin ether (DHE) per mL. Aliquots of this mixture were then held in the dark or irradiated in a flow cell illuminated at a light energy density of 5 J per cm2 provided by a xenon light source equipped with a 630 +/- 5 nm band-pass interference filter; the aliquots were subsequently placed in A.301 cells. All infected cultures were assessed for reverse transcriptase (RT) activity for 17 days. RT activity for either concentration of dye was significantly reduced in irradiated samples as compared to that in samples held in the dark. Blood samples from volunteers also were assessed for the effects of the inactivation process on red cells at concentrations of DHE up to 200 micrograms per mL. No effects were observed on red cell 2,3 DPG or ATP, whole blood potassium concentrations, red cell osmotic fragility, or blood cell antigens.

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.

Synthetic peptides corresponding to sequences in HIV envelope gp41 and gp120 enhance in vitro production of interleukin-1 and tumor necrosis factor but depress production of interferon-alpha, interferon-gamma and interleukin-2.

Patients with human immunodeficiency virus (HIV) infections have aberrant production of a number of lymphokines and monokines. Envelope glycoproteins are believed to be important in HIV pathogenesis and may influence the production of these cytokines. Therefore, synthetic peptides corresponding to amino acid sequences 735-752 and 846-860 of glycoprotein gp41 and to amino acid sequence 304-328 of gp120 were investigated for their abilities to affect the production of the following cytokines by normal peripheral blood mononuclear cells in the presence of appropriate inducers: interferon (IFN)-alpha, IFN-gamma, interleukin (IL)-1, IL-2, and tumor necrosis factor (TNF). In contrast to cells and inducers alone (or in the presence of a control peptide), gp41 or gp120 synthetic peptides were able to depress the production of IFN-alpha, IFN-gamma and IL-2. In contrast, these peptides produced an elevation of the production of IL-1 and TNF. The effect of the gp41 peptides was more marked than that of gp120 peptides in most cases. These studies indicate that these HIV envelope glycoproteins may be directly responsible for aberrant lymphokine and monokine production in patients infected with this virus and therefore may be at least partially responsible for the pathogenesis of AIDS.