The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits.

Although Freund's adjuvant has been used for decades as an immune enhancer in rabbits, adverse physiologic side effects have prompted the search for more suitable alternatives. We used osteocalcin, a bovine bone protein (M.W. 5,800), as the test antigen to evaluate four adjuvant regimens: a) primary inoculation with complete Freund's adjuvant (CFA) followed by three boosts with incomplete Freund's adjuvant (IFA), b) four serial inoculations with RIBI MPL+TDM+CWS adjuvant, c) four serial inoculations with TiterMax #R-1, and d) primary inoculation (only) with TiterMax #R-1. The antibody yield associated with the CFA/IFA regimen (mean OD = 2.152) was at least sixfold that of either TiterMax (mean OD = 0.358) or RIBI (mean OD = 0.239) multiple injection regimens. No antibody response was observed after the single injection of TiterMax antigen emulsion. Maximal antibody production occurred rapidly in response to Freund's adjuvant (day 31) as compared with TiterMax (day 74) and RIBI (day 66).     

T cell tolerance in the chicken. I. Parameters affecting tolerance induction to human gamma-globulin in agammaglobulinemic and normal chickens.

T cell-mediated delayed hypersensitivity (DH) to human gamma-globulin (HGG) can be induced in chickens by subcutaneous injection of the antigen in complete Freund's adjuvant (CFA). In the present work, it has been demonstrated that specific tolerance of the cells mediating this DH can readily be induced in both normal and bursectomized (BX) FP strain chickens by simple i.v. injection of soluble antigen, regardless of the presence of antibody production to the tolerogen. A significant degree of tolerance at the DH and helper T cell levels could be generated in BX birds by injection of as little as 0.5 mg of HGG; such a dose only induced tolerance in normal birds when it had been previously deagregated by ultracentrifugation. Regular, nondeaggregated antigen could produce tolerance in normal animals, but only at doses of greater than 5 mg. The tolerizing injection induced a primary antibody response in normal birds in all cases, but a secondary response could not be obtained in animals rendered tolerant at the T cell level. Establishment of tolerance appeared to be very rapid, and animals remained refractory to induction of DH for at least 3 weeks after the tolerizing injection. The mode in which the antigen was presented to the animals appeared to be crucial in determining whether tolerance or sensitivity would be established.     

A comparison of antibody titres in mouse uterine fluid after immunization by several routes, and the effect of the uterus on antibody titres in vaginal fluid

Measurements of specific antibody titres in uterine fluid of mice immunized by different routes indicated that two immunizations in the pelvic presacral space using aluminium hydroxide as adjuvant was a simple and effective way to elicit a significant IgA and IgG response. Higher IgA and IgG titres were produced in uterine fluid by subcutaneous immunization with antigen in Freund's complete adjuvant followed by intravaginal boosting without adjuvant, but this immunization involved both a toxic adjuvant and repeated applications of large doses of antigen in the vagina. Intragastric immunization produced an IgA response in the uterus but no IgG. Local intravaginal priming and boosting with large doses of antigen without adjuvant produced an IgA response in uterine fluid, but was less effective for IgG and was inefficient in terms of time and the amount of antigen used. Hysterectomy reduced the concentration of specific IgA in vaginal fluid of immunized mice to no more than 5% of normal, indicating that most of the IgA in vaginal fluid originates in the uterus. In contrast, IgG titres were not significantly different in hysterectomized and intact mice. IgA titres in vaginal fluid were at least partly restored to normal levels in sham-hysterectomized mice.     

Characterization of antigens and antibody specificities involved in Heymann nephritis

The present study was conducted to determine if Fx1A, a renal cortical extract used to induce Heymann nephritis, contains nephritogenic antigens in addition to the brush border-derived glycoprotein gp 330. Of 26 Lewis rats immunized with Fx1A, 24 developed abnormal proteinuria (greater than 20 mg/24 hr) by wk 10, whereas of 15 rats immunized with a partially purified gp 330 preparation (MVH), only one developed proteinuria. Immunofluorescence studies showed that all Fx1A rats developed large, diffuse, granular deposits along the glomerular basement membrane which stained brightly for IgG and C3; only 11 of the 15 MVH rats had definite deposits; in most rats, they were small and stained only moderately for IgG and faintly or not at all for C3. The Fx1A and MVH rats developed comparable levels of antibodies to MVH (gp 330) before the onset of proteinuria in Fx1A rats, after which serum IgG and antibody levels declined. In contrast, antibodies against soluble Fx1A antigens appeared earlier and rose more rapidly in Fx1A than in MVH rats. Larger amounts of IgG could be eluted from the glomeruli of Fx1A rats than from MVH rats. Eluates from the Fx1A rats contained antibodies that reacted with gp 330 and also a 95 kd antigen; the latter reactivity was not demonstrated in eluates of MVH rats. Immunoprecipitation studies showed that both gp 330 and the 95 kd antigen are components of normal glomeruli. The results show that immunization with Fx1A produces a more severe form of Heymann nephritis than does gp 330, and that Fx1A contains at least one nephritogenic antigen in addition to gp 330.     

Immunogenic Properties of Colloidal Gold

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or its combination with Freund's adjuvant). Application of colloidal gold also enhanced nonspecific immune responses, such as lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens or complete antigens (without other adjuvants) was shown to induce the production of highly active antibodies. In addition, the amount of antigen used for animal immunization with colloidal gold was an order of magnitude lower, compared to immunization with complete Freund's adjuvant. This fact can be evidence for adjuvant properties of colloidal gold proper.     

Immune response in rats and mice with adjuvant disease

There were phasic changes of immune response in rats and mice with adjuvant disease: an increase of antibody production on the 7th day after the injection of complete Freund's adjuvant and its depression on the 21st day; there was also observed suppression of normal antibody production to O- and Vi-antigens of typhoid bacilli.     

Definition of the pulmonary antibody response to ovalbumin following local challenge in systemically immunized rats

The in vivo pulmonary immune response of rats to local stimulation with antigen was assessed by measuring antigen-specific antibody and antibody-secreting cells utilizing enzyme-immunoassay technology. Sprague-Dawley rats were immunized subcutaneously with ovalbumin (OA) emulsified in Freund's incomplete adjuvant, challenged with OA intratracheally on Day 19 and sacrificed 1, 2, 3, or 4 days later. Specific antibody-secreting cells in the lung-associated lymph nodes were enumerated with the ELISA-SPOT assay and antibody concentration in the pulmonary lavage fluids and sera was assessed with the ELISA. The greatest response for each parameter was on Day 2. Cellular infiltration of the lung was minimal. Cellular infiltrates consisted mainly of polymorphonuclear leukocytes and were most numerous in the lavage fluid on Days 1 and 2 and in the lung parenchyma on Day 2 after challenge. Local production versus serum transudation of antibody was evaluated by comparing the levels of OA-specific antibody to albumin in the lavage fluid and serum. The data showed that antibody in the lungs was primarily produced locally.     

Vaccines against blowfly strike: the effect of adjuvant type on vaccine effectiveness.

Vaccination of sheep with a partially purified extract of Lucilia cuprina larvae in some cases resulted in marked reduction of growth in larvae which fed on the sheep. Twelve adjuvants were assessed, in vitro and in vivo, to determine which induced the largest inhibitory effect on larval growth. The Freund's complete adjuvant and Quil A groups produced ELISA antibody levels significantly higher (P less than 0.05) than other groups. Seven adjuvants mediated an immune response which caused significant inhibition of larval growth (P less than 0.05). When the sheep were assessed by in vivo larval culture, only larvae feeding on sheep vaccinated with the antigen presented in Freund's complete adjuvant or dextran sulphate or a dextran sulphate/Freund's incomplete adjuvant mixture weighed significantly less (P less than 0.05) than larvae feeding on control sheep. The effect on larvae was monitored in vitro for 70 days after vaccination, by which time significant reduction in larval weight was no longer observed. The loss of larval growth inhibition was not associated with a corresponding reduction in overall antibody levels.     

An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits.

Emulsion adjuvants have been used for production of polyclonal antisera in rabbits (Oryctolagus cunniculi) for decades. Complete Freund's adjuvant has a reputation as a very effective immunoenhancer, but adverse physiological effects, including fever, inflammation and sterile abscess formation, have prompted a search for alternatives to complete Freund's. In this study, we quantitatively compared five adjuvant regimens: (a) a primary inoculation with complete Freund's followed by three boosts with incomplete Freund's; (b) four serial inoculations of incomplete Freund's adjuvant augmented with 6-bromoguanisine; (c) four serial inoculations with RIBI's MPL + TDM + CWS adjuvant emulsion; (d) four serial inoculations with Montanide ISA 50 emulsion; and (e) four serial inoculations with Montanide ISA 70 emulsion. We chose a small (12 amino acid) chain polypeptide coupled to bovine serum albumin as our test antigen. When compared, no system could be seen to be significantly better than a regimen of a primary immunization with complete Freund's adjuvant followed by serial reimmunization with incomplete Freund's adjuvant. The commercially available RIBI adjuvant produced significantly lower antibody levels, while other systems produced essentially equivalent levels. With all five adjuvants, antibody quantities plateaued after the second injection and further immunization did not increase titers significantly. Boost injections did yield greater intradermal tissue reaction than primary inoculations, and intramuscular inoculum volumes of 0.4 cc caused chronic lesions still detectable by the gross necropsy 2 weeks after the final injection.     

Determinant specific suppression of antigen-induced T cell proliferation in the guinea pig. II. Determinant specific suppression of in vitro T cell responsiveness parallels a selective suppression of anti-hapten but not anti-carrier antibody responses

Using an antigen of defined physical structure with precisely mapped immunogenic sites, we asked whether those molecular sites previously shown to be critical for immune response gene-mediated initiation of T cell proliferation and T help correspond to the same molecular regions capable of inducing antigen-specific suppression of T cell proliferation and antibody production. Inbred strain 2, 13, and 2 x 13 F1 hybrid guinea pigs were immunized with various species variants or fragments of insulin adjuvant before subsequent immunization with antigen in complete Freund's adjuvant. Analysis of the patterns of depressed T cell responsiveness showed a striking correspondence to the Ir gene-dependent mechanism that controls the recognition of discrete regions within the insulin molecule observed in T cell help in antibody production. In addition, suppression of carrier-specific T cells parallels suppression of anti-hapten antibody responses when hapten is presented on the suppressed carrier without a concomitant suppression of the anti-carrier antibody response.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Immunoregulation of experimental allergic encephalomyelitis: conditions for induction of suppressor cells and analysis of mechanism.

We determined requirements for the induction of immunoregulatory suppressor cells in experimental allergic encephalomyelitis (EAE) in Lewis rats. Pretreatment of rats with myelin basic protein (BP) in incomplete Freund's adjuvant (IFA) stimulates the proliferation of suppressor cells that localize in lymph nodes and spleen (but not thymus) and exert control over the development of clinical EAE. Dosage studies revealed that 3 X 10(7) suppressor cells can adoptively transfer suppression to syngeneic recipients. Transferred unresponsiveness wanes within 3 weeks, indicating that the suppressor cells are short-lived lymphocytes, although actively induced unresponsiveness persists for at least 8 weeks, probably as a result of continual proliferation under the influence of antigen. No evidence was obtained to suggest that antigen carry-over or blocking antibody production accounts for adoptive transfer of unresponsiveness. Suppressor cells apparently act at the inductive phase of the immune response since they had no inhibitory effect on adoptive transfer of disease by effector lymph node cells. Other mechanisms also may play a role in unresponsiveness to EAE, since rats pretreated i.v. with high dosages of soluble BP were temporarily rendered unresponsive, although suppressor cells could not be detected in these animals.     

An evaluation of distress following intraperitoneal immunization with Freund's adjuvant in mice

Intraperitoneal immunization with Freund's adjuvant is frequently used to stimulate antibody production in mice. To evaluate the clinical and pathological effects of this technique, mice were immunized intraperitoneally with complete Freund's adjuvant and albumin, and the injection repeated 3-4 weeks later using incomplete Freund's adjuvant. This regimen induced a mean antibody titer against albumin of 1:280 within 7 days after booster immunization and increased the abdominal width, abdominal circumference and spleen weights of immunized animals. Food intake and body weight decreased after immunization, but returned to control levels within 1-2 weeks. Open-field activity was not affected. Neutrophilia, eosinophilia and monocytosis were present 7 days after immunization and persisted for the duration of the study. Gross and histopathological lesions included multiple granulomatous abdominal adhesions and lymphoid hyperplasia. Thus, intraperitoneal immunization with Freund's adjuvant and albumin produced some adverse effects in the animal (weight loss, neutrophilia and granulomatous peritonitis). However, the animals did not appear to be severely or chronically impaired, since food intake, body weight and locomotor activity were within normal limits for most of the post-immunization period.     

Attenuation of collagen arthritis and modulation of delayed-type hypersensitivity by type II collagen reactive T-cell lines

T-cell lines were established from the lymph node cells of syngeneic Louvain (LOU) rats previously immunized with native chick type II collagen (CII) emulsified in incomplete Freund's adjuvant. The CII lines proliferated in vitro to type II collagen but not to type I collagen, ovalbumin (OV), or PPD. Control lines, developed from LOU rats immunized with OV emulsified in complete Freund's adjuvant, were OV specific because they did not respond to other antigens in vitro. CII line cells could adoptively transfer delayed-type hypersensitivity (DTH) but did not induce IgG antibody production to collagen. Moreover, the intravenous administration of 2 X 10(7) CII line cells prevented the subsequent induction of collagen arthritis following immunization and suppressed DTH to collagen without affecting antibody responses in the recipients. Spleen cells, but not sera, from these resistant rats decreased CII line reactivity in vitro. OV or irradiated CII lines had no effect on clinical or immunologic parameters in this model. These findings demonstrate protection from arthritis afforded by T-cell line transfer and suggest that the phenomenon results from down-regulation of the recipients' cellular immunity to collagen.     

Evaluation of ethylene-vinyl acetate copolymer as a non-inflammatory alternative to Freund's complete adjuvant in rabbits

Ethylene-vinyl acetate copolymer (EVAc) was evaluated as an antigen delivery device in laboratory rabbits. Bovine serum albumin (BSA) was incorporated with EVAc in a pellet, which was implanted subcutaneously. Serum antibody titers to BSA in four implanted rabbits were equal to titers in four rabbits injected twice with BSA in complete Freund's adjuvant. Three of four rabbits implanted with EVAc displayed no inflammation or systemic illness in response to the pellet. The fourth rabbit repeatedly developed a small abscess at the implantation site, but the lesions were less severe than complete Freund's adjuvant injection sites. The EVAc pellet is recommended as a non-inflammatory alternative method to Freund's adjuvants for producing serum antibody in rabbits.     

Modification of immune reactions by antigen-immunosuppressive agent conjugates. V. Studies on antigen-specific activity of 6-mercaptopurine bovine gamma globulin conjugates on the humoral immune response in rabbits]

Rabbits treated daily and for seven consecutive days with 6-mercaptopurine bovine gamma globulin conjugates (MPI--n-BGG; 'I'--characterizes the king of chemical binding and 'n' the number of coupled MP-residues per one mole BGG) show an altered immunological reactivity. A following intradermal immunization with BGG alum adsorbate results in a suppressed anti BGG antibody production on the third day after antigen application (antibody titer 1:320, antibody titer of control animals--pretreated with BGG and uncoupled 6-MP equals 1:5120). Already three days later the antibody titers of the test groups show a significant increase and are two dilution stages higher than the titers of the controls. A suppressive effect on the third day is induced by MPI--13-BGG, MPI--26-BGG, MPI--36-BGG and MPI--46-BGG; the later adjuvant effect can only be seen in the MPI--26-BGG, MPI--36-BGG and MPI--46-BGG but not in the MPI--13-BGG pretreated animal group. While the short time suppression was antigen specific--the humoral immune response against a second unrelated antigen was not reduced--the adjuvans effect was not antigen specific. A pretreatment with the substances mentioned above results in an increased anti BGG and anti HSA serum antibody level. Comparing investigations on the unspecific immunosuppression in rabbits by 6-mercaptopurine shows that application of 10 mg 6-MP/kg/day for seven days at first leads to a suppression but later on to an enhancement of antibody production. Application of 10 mg 6-MP/kg/day for 10 days results in a long lasting suppression without enhancement effect. As a reason for these differences the different catabolism of immunosuppressive agent and antigen is discussed. For the phenomena following antigen specific immunosuppression, similar mechanisms can be responsible.     

Stimulation of the in vivo dinitrophenyl antibody response to the DNP conjugate of L-glutamic acid60-L-alanine30-L-Tyrosine10 (GAT) polymer by a synthetic adjuvant, muramyl dipeptide (MDP): target cells for adjuvant activity and isotypic pattern of MDP-stimulated response

Specific anti-dinitrophenyl (DNP) response to DNP-conjugated L-glutamine60-L-alanine30-L-tyrosine10 (DNP-GAT) was obtained in GAT-responder mice by using synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) as adjuvant. Significant levels of anti-DNP antibodies were observed during a secondary response to DNP-GAT, when both antigen and MDP were used for priming. In this system, MDP was able to prime the carrier-specific T cells but not the hapten specific B cells. The study of the isotypic pattern of the anti-DNP response shows that MDP stimulates only the appearance of specific anti-DNP IgG1 plaque-forming cells. Anti-DNP plaque-forming cells were stimulated in animals primed with DNP-GAT in Freund's complete adjuvant or in Maalox-pertussis and used as control IgG1, IgG2a, and IgG2b.     

Immunoregulation in experimental interstitial nephritis: immunization with renal tubular antigen in incomplete Freund's adjuvant induces major histocompatibility complex-restricted, OX8+ suppressor T cells which are antigen-specific and inhibit the expression of disease

We utilized a model of experimental interstitial nephritis induced by renal tubular antigen in complete Freund's adjuvant to examine a mechanism of immunologic tolerance produced by priming immunization with tubular antigen in incomplete Freund's adjuvant. Brown Norway rats primed with tubular antigen in incomplete adjuvant do not develop significant nephritis after challenge with antigen in complete adjuvant, and this tolerance can be transferred to naive recipients with donor T cells. These T cells also specifically suppress a delayed-type hypersensitivity response to soluble tubular antigen in recipients immunized to produce disease. This suppression is MHC-restricted and is mediated by OX8+ T cells which bind antigen and bear idiotypes cross-reactive with those on antibodies eluted from the tubular basement membrane. Despite the suppression of histologic disease, tolerized animals were able to produce significant titers of antibodies to tubular basement membrane. Our findings demonstrate an additional strategy for altering the natural history of immune-mediated renal disease, and further refine the characterization of the suppressive effect produced by incomplete Freund's adjuvant.     

IgE-selective and antigen-specific unresponsiveness in mice. I. Induction of the unresponsiveness by administration of ovalbumin-pullulan conjugate.

An experimental model was demonstrated in mice for the induction of IgE-selective unresponsiveness to ovalbumin, a protein antigen. An administration of ovalbumin, conjugated with pullulan, a linear polymer of glucose, (OA-pullulan) into mice resulted in the induction of a long lasting, IgE-selective unresponsiveness to the subsequent immunization with native OA in the form optimal to elicit IgE antibody response. The IgE-selective unresponsiveness is antigen specific and is infectious to normal mice by transferring the spleen cells from mice receiving OA-pullulan conjugate at least 2 weeks before. In contrast to other modified antigens, OA-pullulan was found to elicit good IgM and IgG antibody responses, but not an IgE response, without the aid of an adjuvant.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.